Design,Synthesis And Preliminary Bio-Evaluation Of Anti-Cancer Drugs Targeting Class Ⅰ Histone Deacetylases

The acetylation and deacetylation of histones are critical to the modification of chromatin structure,and it is also one of the most widely studied epigenetic modifications.The acetylation status of histones is regulated by two groups of enzymeshistone deacetylases(HDACs)and histone methyltransferases(HATs).In addition,more and more non-histone proteins have also been identified as substrates of HDACs,including proteins related to transcription complexes,which have profound effects on the regulation of gene expression,cell proliferation,migration,death,and angiogenesis.According to the homology difference of sequence in the germline,subcellular localization and enzyme activity,18 known human HDACs can be divided into four subfamilies,among which the class I HDACs are most closely related to the oncogenesis and development of cancer.Class I HDACs can promote cancer cell proliferation,invasion and migration,induce cancer tissue angiogenesis,enhance cancer cell resistance to chemotherapy drugs,and inhibit differentiation and apoptosis of cancer cells.Therefore,HDACs have become an important target in the current field of anti-tumor drug research.At present,many HDACs inhibitors have been launched in China and other countries,and more than 20 HDACs inhibitors were evaluated in preclinical or clinical trials,which strongly proves the feasibility and effectiveness of HDACs inhibitors as anticancer drugs.According to their chemical structures,HDACs inhibitors can be divided into hydroxamic acids,carboxylic acids,aminobenzamides,cyclic peptides,etc.Most HDACs inhibitors are mainly composed of the following four parts:surface recognition motif,polar connection unit(CU),linker and Zn2+chelating group(ZBG).Among them,the Zn2+chelating group is the key part of the inhibitors,and the role to chelate the zinc ion at the bottom of the catalytic pocket of HDACs enzyme.However,HDACs inhibitors showed poor anti-tumor activity against various solid tumors in preclinical and clinical trials,which severely limits the application.HDACs inhibitors are often used in combination in clinical applications,but the ubiquitous hydroxamic acid in the structure makes it metabolized faster,and it is difficult to combine with other drugs.Because the catalytic pocket of HDACs has a certain degree of conservation,many HDACs inhibitors have poor selectivity and inhibit the subtypes that are not related to tumors,which causes side effects.In order to solve the above problems,two series of quinoline derivatives were designed,synthesized as class I HDACs inhibitors in Chapter 2.Among them,the series I target compounds were designed according to the structure-activity relationship and molecular docking results in our previous studies,which include introducing different substitutions as cap group or linker and ZBG at 4 or 2 position of quinoline.The preliminary biological evaluation suggested that the most active hydroxamic acid target compound HI-6 and o-phenylenediamide target compound HI-29 have HDAC1 inhibitory activity of 0.38 nM and 87.54 nM,respectively,which are significantly better than the positive controls SAHA and MS-275.In the whole cell HDACs selectivity experiments,the two target compounds showed good class I HDACs selectivity.In addition,the results of anti-tumor cell proliferation indicated that HI-6 and HI-29 were more potent than the positive control drugs.At the same time,HI-6 and HI-29 can induce tumor cells apoptosis as well as autophagy,and also inhibit the invasion and migration of breast cancer cells.In the druggability evaluation,the two target compounds are less toxic to normal cells,and their metabolic properties are significantly better than the positive control drugs SAHA and MS-275.This work is an important attempt to develop class I HDACs inhibitors with quinoline as the core of structures.The relevant target compounds have good biological activity and druggability,and have the potential for further in-depth research and development.As a member of class I HDACs,there are relatively few studies on HD AC 8 inhibitors,but our group constructed a ligand-based pharmacophore model in our previous studies to provide a powerful tool for the discovery of HDAC8 inhibitors.In order to develop selective inhibitors that may have HDAC8 inhibitory activity in class I HDACs,series Ⅱ target compounds were designed and synthesized,and 4hydroxyquinoline-2-carboxylic acid(Kynurenic acid)was also used as the core.Different substituents at 4 position were introduced as the enzyme surface recognition region and target compounds formed an L-shaped conformation to meet the ligandbased structure pharmacophore model constructed in the previous work of our group.By changing different enzyme surface recognition regions and zinc ion chelating groups,related structure-activity relationships were explored in order to find selective inhibitors with HDAC8 inhibitory activity.In the bioactivity evaluation,the series Ⅱ target compound HE-2 showed micromolar HDAC8 activity,which was more potent than the positive drug SAHA.In the anti-tumor cell proliferation activity experiments,the target compounds HE-2 and HE-3 showed good activity,significantly better than the positive control PCI-34051(an HDAC8 selective inhibitor).This work is an exploration of the development of selective inhibitors of HDAC8 with quinoline as core structures.Related active compounds can be used as lead compounds for further structural modification.In order to solve the problems of side effects and complex metabolism of HDACs inhibitors-based combinations,in Chapter 3,HDACs and proteasome dual-target inhibitors were designed and synthesized.By integrating the active structure of the proteasome into the pharmacophore of HDACs inhibitors,the target compound has dual target inhibitory activity,which could solve the problems of proteasome inhibitor resistance and inconsistent pharmacokinetic properties of multi-drug combination.The ubiquitin-proteasome system(UPS)is an ATP-dependent degradation system related to protein quality control and cell homeostasis maintenance in eukaryotic cells,which can specifically recognize and degrade a variety of protein substrates,especially unstable or denatured,misfolded proteins.Proteasome inhibitors selectively induce tumor cell apoptosis and improve sensitivity to radiotherapy and chemotherapy.However,clinically,the proteasome inhibitor bortezomib alone will almost inevitably lead to drug resistance and relapse.For this reason,exploring the best combination of bortezomib and other anticancer drugs is one of the hot spots in the current clinical research concern.HDACs inhibitors have significant selective anti-tumor effects by inhibiting HDACs and changing tumor genome expression patterns.Many studies on their combination with proteasome inhibitors have entered the clinical stage.Among them,the combination of panobinostat and bortezomib has been approved by the FDA for the treatment of relapsed and drug-resistant multiple myeloma.However,due to poor patient compliance,complex pharmacokinetics and challenges related to drug interactions,a single drug combination may not be the best strategy.On the contrary,multi-target drugs may help solve these problems.Therefore,in this chapter,the common pharmacophores of HDA C inhibitors are incorporated into the solvent exposed part of bortezomib,and hydroxamic acid derivatives or o-aminobenzamide derivatives were designed as histone deacetylases and proteasome dual inhibitors.By introducing different zinc-binding groups into the peptide boronate and changing the amino acid residues,the structure-activity relationships of the new dual-target inhibitors were studied.The most effective inhibitor ZY-2 has excellent proteasome inhibition activity and has good selectivity for class I histone deacetylase HDAC1(IC50 values are 1.1 nM and 255 nM,respectively).In particular,ZY-2 not only shows good antiproliferative activity against multiple myeloma cell lines RPMI-8226,U266,KM3 and NCI-H929(IC50 values are 6.66 nM,4.31 nM,10.1 nM and 1.75 nM,respectively).Moreover,compared with bortezomib,ZY-2 has more effective anti-proliferative activity(IC50 value 8.98 nM vs.226 nM,P<0.01)on the multiple myeloma cell line KM3/BTZ,which is resistant to bortezomib.It is even better than the combination of class I HDACs inhibitor MS-275 and bortezomib(1:1)(IC50 value 8.98 nM vs.98.0 nM,P<0.01).As far as we were concerned,this is the first attempt to design and synthesize a covalent proteasome inhibitor with HDACs inhibitory activity.It is also the first exploration to design class I HDACs and proteasome dual-target inhibitors for the treatment of proteasome inhibitors-resistant multiple myeloma.In summary,this thesis focused on the problems of HDACs inhibitors.Through computer-aided drug design and target-based drug design,two categories of small molecule anti-tumor drugs based on class I HDACs are designed and synthesized.A preliminary activity evaluation was carried out.In Chapter 2,thirty-five series I target compounds and twenty-three series II target compounds were designed and synthesized.The series I target compounds were served as class I HDACs inhibitors.In the in vitro evaluation,they showed good enzyme activity,anti-tumor activity as well as good druggability and can be used as potential drug candidates or leads for more in-depth evaluation and modification.Series II target compounds as selective inhibitors of HDAC8,a member of class I HDACs,exhibit certain enzymes inhibition and antitumor activity and also can be regarded as lead compounds for further studies.In Chapter 3,fourteen dual inhibitors of HDACs and proteasome were designed and synthesized.Among them,ZY-2 and ZY-13 have good dual-target inhibitory activity and good performance in anti-multiple myeloma activity in vitro experiments.And especially,they have a significant anti-drug resistance effect on bortezomib-resistant multiple myeloma cells.